Related Applications
Subfertility is a major problem with domestic animals, humans and certain endangered species. Subfertility has a billion dollar impact on food production. In many circumstances, most sperm in a sample must have a low fertilizing potential despite normality of motion, morphology, or "viability" (plasma membrane excludes dye) . This is evidenced in two ways. First, attributes of each spermatozoan other than "motility" and "viability" are important, and failure in one of many critical functions renders a spermatozoan incapable of fertilizing an oocyte (Amann and Hammerstedt, 1993; Hammerstedt, 1996). An in vitro sperm-binding assay (Barbato et al., 1998; Amann et al., 1999) allows detection of certain males ejaculating semen with a relatively low percentage of sperm capable of binding in this assay and, hence, likely to be of low fertility. Fertility trials confirm general correctness of the classification (Barbato et al., 1998).
Knowledge of sperm-associated proteins is evolving rapidly, and it is evident that sperm are exposed to, and modified by, specific proteins secreted at multiple sites within the testis and/or excurrent duct system. As summarized by Amann et al. (1999), individual glycosylated molecules have received emphasis in considerations of sperm-to-egg binding, at the potential risk of overlooking non-glycosylated molecules. Also, some authors rightly have highlighted the need for multiple and synergistic ligands. Hammerstedt et al. (1997) disclosed a novel native protein and several non-glycosylated, synthetic peptides which, when included in buffer suspending sperm, increased the percentage of sperm bound using an in vitro assay and also increased fertility of sperm used for artificial insemination. The native peptide is thought to be formed from prosaposin.
Full-length, unprocessed prosaposin (Collard et al., 1988; O'Brien and Kishimoto, 1991; Kishimoto et al., 1992; Azuma et al., 1998) also has been termed sulfated glycoprotein 1 or SGP-1. Conventionally, prosaposin is proteolytically processed (Hiraiwa et al., 1993) into 4 saposins (A, B, C and D; each 13 to 15 kD) and 3 intervening segments (A-B, B-C and C-D; each 6 to 8 kD). Saposins are considered to be activator proteins increasing the catalytic rate of lipid-modifying enzymes, as in lysosomes (O'Brien and Kishimoto, 1991; Kishimoto et al., 1992; Mumford et al., 1995). Until Hammerstedt et al. (1997) , no function had been ascribed to any of the 3 intervening segments. Prosaposin (or mature products thereof) is produced throughout the body although not secreted by most tissues (Collard et al., 1988; Kishimoto et al., 1992; Sylvester et al., 1984, 1989; Igdoura et al., 1993; Rosenthal et al., 1995).
The antibody commonly used to localize prosaposin apparently binds an epitope(s) common to each of the 4 saposins (Igdoura et al., 1993; Igdoura and Morales, 1995) and, hence, also is bound by prosaposin. Based on probing of western blots with antibody, prosaposin (and traces of saposins) was found in luminal fluids in the excurrent ducts conveying sperm from the testes (Sylvester et al., 1989; Igdoura and Morales, 1995), but there was a complete absence of sperm-labeling throughout the excurrent ducts (Sylvester et al., 1989; Hermo et al., 1992).
The most favored synthetic peptides of Hammerstedt et al. (1997) are a sequence integral to a truncated portion of the rat or human prosaposin molecule extending from the cystine amino acid near the distal terminus of saposin A, through the intervening sequence between saposin A and saposin B., and through the proximal cystine amino acid in saposin B; a total of sixty (60) amino acids in rat or sixty-one (61) amino acids in human. Hammerstedt et al. (1997) further claim that maximum activity is obtained by linking the terminal cystine amino acids in most favored disclosed sequences by a disulfide bond, to provide a hairpin form.